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Method for the recovery of non-segmented, nagative-stranded rna viruses from cdna

An RNA virus, segmented technology, applied in the field of improvement of the recovery of non-segmented negative-strand RNA viruses from cDNA, which can solve problems such as limiting the number of T7 cell lines

Inactive Publication Date: 2006-11-29
WYETH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This greatly limits the number of T7 cell lines available for rescue and poses serious problems for the generation of competent cell lines for many non-segmented negative-sense RNA viruses

Method used

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  • Method for the recovery of non-segmented, nagative-stranded rna viruses from cdna
  • Method for the recovery of non-segmented, nagative-stranded rna viruses from cdna
  • Method for the recovery of non-segmented, nagative-stranded rna viruses from cdna

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0229] A Universal Calcium Phosphate Transfection Method for the Rescue of Nonsegmented Negative-Strand RNA Viruses from Vero Cells

[0230] the solution

[0231] The following solutions are typically used for host cell transfection: 280 mM NaCl [16.4 g NaCl (or 56 mL 5M NaCl)], 50 mM BES [10.7 g BES (without acid form)] and 1.5 mM sodium phosphate [0.21 g NaCl 2 HPO 4 ] in 2×BBS (per L) solution (2×BES-buffered saline). The pH of the BBS solution was adjusted to 6.95-6.98 with NaOH. The solution was then filter sterilized and stored frozen.

[0232] Prepare 2.5M CaCl with 36.8 g calcium chloride per 100 ml total volume 2 solution, stored at -20°C. The solution was sterilized by filtration with nitrocellulose (membrane). Avoid cellulose acetate filters as they will clog. Alternatively, autoclave the transfection solution. However, the latter step is less necessary since the 2x BBS solution may be slightly altered during autoclaving.

[0233] The following solutions ar...

Embodiment II

[0245] A Universal Calcium Phosphate Transfection Method to Rescue Paramyxoviruses

[0246] The calcium phosphate transfection procedure used in the following alternative transfection method is based on the method of Chen and Okayama, Mol. Cell. Biol. 7:2745-2752, 1987, which is incorporated herein by reference.

[0247] Exemplary DNA:

[0248] 1. The full-length viral genome cDNA clone contains the T7 promoter fused to the 5' end of the sense strand and the ribozyme fused to the 3' end.

[0249] 2. Protein expression plasmid, controlled by T7 RNA polymerase promoter, encoding nucleocapsid protein (N or NP), phosphoprotein (P) and polymerase protein (L).

[0250] 3. A plasmid encoding the T7 RNA polymerase protein under the control of the immediate early transcriptional control region of human cytomegalovirus.

[0251] Exemplary calcium phosphate transfection reagents:

[0252]1. 2X BES-buffered saline: 50nM BES (pH 6.95-6.98), 280mM NaCl, 1.5mM Na 2 HPO ...

Embodiment III

[0275] Rescue of measles virus (MV) in Vero cells

[0276] Measles virus was rescued in Vero cells using the exemplary transfection method described in Example I above. The source of T7 was the plasmid pSC6-T7 (no MVA) or MVA / T7 (Wyatt et al., 1995). The results indicated successful rescue of many MV(+), as shown in Table 3 below (columns A-F represent different rescue experiments performed on different days, each experiment represented by multiple transfections - up to 12 individual transfections in experiments E and F Dye (hole)).

[0277] table 3

[0278] Rescue of measles virus with plasmid-T7 or MVA / T7

[0279]

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Abstract

Methods for producing infectious, non-segmented, negative-stranded RNA viruses of the Order Mononegavirales are provided that involve coexpression of a viral cDNA along with essential viral proteins, N, P, and L in a host cell transiently transfected with an expression vector encoding an RNA polymerase. In alternate methods, after the host cell is transfected with a viral cDNA expression vector and one or more vectors encoding the RNA polymerase, N protein, P protein, and L protein, the host cell is exposed to an effective heat shock under conditions sufficient to increase recovery of the recombinant virus. In other alternate embodiments, the host cells are transferred after viral rescue begins into co-culture with a plaque expansion cell, typically a monolayer of expansion cells, and the assembled infectious, non-segmented, negative-stranded RNA virus is recovered from the co-culture. Also provided within the invention are compositions for producing infectious, non-segmented, negative-stranded RNA virus of the Order Mononegavirales, recombinant viruses produced using the foregoing methods and compositions, and immunogenic compositions and methods employing the recombinant viruses. In additional embodiments, the methods and compositions of the invention are employed to produce growth- or replication-defective non-segmented negative-stranded RNA viruses and subviral particles.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application 60 / 477,389, filed June 9, 2003, which is incorporated herein by reference. field of invention [0003] The present invention relates to improved methods and compositions for producing infectious, replicating, non-segmented negative-strand RNA viruses of the order known as mononegavirales. The methods and compositions are also suitable for producing growth or replication deficient of these viruses. Background of the invention [0004] There are enveloped, non-segmented, negative-sense, single-stranded RNA viruses unique in composition and expression. The genomic RNA of negative-sense, single-stranded viruses has two template functions within the nucleocapsid content: as a template for the synthesis of messenger RNA (mRNA) and as a template for the synthesis of the antisense genomic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86A61K39/12C12N7/00A61K48/00C07K14/115C07K14/12C07K14/145C12N5/10C12N7/04
CPCA61K2039/5256C12N2760/18551C12N2760/18422A61K39/12A61K39/205C12N7/00C12N2760/18022C07K14/005C12N2760/20262C12N2760/18622A61K2039/5258A61K48/00C12N2760/18652C12N2760/20251C12N15/86C12N2760/18634C12N2760/20234A61K39/155A61K39/165C12N2760/18534C12N2760/18452C12N2760/18552C12N2760/20243C12N2760/18662C12N2760/18651A61K2039/5254C12N2760/18434C12N2760/20252C12N2760/18451A61K39/00A61P11/00C12N15/10
Inventor C·L·派克斯S·A·乌登M·S·西德胡
Owner WYETH LLC
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